Crystallized P38 complexes

ABSTRACT

This invention provides certain crystallized, protein kinase-ligand complexes, in particular P38-ligand complexes, and their structure coordinates. The structure coordinates are based on the structure of a phosphorylated P38γ complex which has now been solved and which reveals new structural information useful for understanding the activated states of other, related kinase proteins as described herein. The key structural features of the proteins, particularly the shape of the substrate binding site, are useful in methods for designing or identifying selective inhibitors of the protein kinases, particularly P38γ and in solving the structures of other proteins with similar features. The structure coordinates may be encoded in a data storage medium for use with a computer for graphical three-dimensional representation of the structure and for computer-aided molecular design of new inhibitors.

TECHNICAL FIELD OF INVENTION

[0001] This application claims priority from U.S. ProvisionalApplications Serial No. 60/112,354 filed Dec. 16, 1998, and U.S.Provisional Application Serial No. 60/163,373 filed Nov. 3, 1999.

[0002] This invention relates to certain crystallized kinaseprotein-ligand complexes, particularly complexes of crystallized P38protein, and more particularly complexes of P38γ protein. This inventionalso relates to crystallizable compositions from which theprotein-ligand complexes may be obtained. This invention also relates tocomputational methods of using structure coordinates of the proteincomplex to screen for and design compounds that interact with theprotein, particularly P38 protein or homologues thereof.

BACKGROUND OF THE INVENTION

[0003] Mammalian cells respond to extracellular stimuli by activatingsignaling cascades that are mediated by members of the mitogen-activatedprotein (MAP) kinase family. Mammalian mitogen-activated protein (MAP)kinases are proline-directed serine/threonine kinases that facilitatesignal translocation in cells [Davis, Mol. Reprod. Dev. 42, 459-467(1995); Cobb et al., J. Biol. Chem. 270, 14843-14846 (1995); Marshall,Cell 80, 179-185 (1995)]. MAP kinases include the extracellular-signalregulated kinases (ERKs), the c-Jun NH₂-terminal kinases (JNKs) and theP38 kinases, which have similar sequences and three-dimensionalstructures [Taylor & lipopolysaccharides (LPS), UV, anisomycin, orosmotic shock, and by cytokines, such as interleukin-1 (IL-1) and tissuenecrosis factor (TNF). Inhibition of P38α kinase leads to a blockade onthe production of both IL-1 and TNF. IL-1 and TNF stimulate theproduction of other proinflammatory cytokines such as IL-6 and IL-8 andhave been implicated in acute and chronic inflammatory diseases and inpost-menopausal osteoporosis [Kimble et al., Endocrinol., 136, 3054-61(1995)].

[0004] Based upon this finding it is believed that P38α, along withother MAPKs, has a role in mediating cellular response to inflammatorystimuli, such as leukocyte accumulation, macrophage/monocyte activation,tissue resorption, fever, acute phase responses and neutrophilia. Inaddition, the MAPKs, such as P38α, have been implicated in cancer,thrombin-induced platelet aggregation, immunodeficiency disorders,autoimmune diseases, cell death, allergies, osteoporosis andneurodegenerative disorders. Inhibitors of P38α also appear to beinvolved in pain management through inhibition of prostaglandinendoperoxide synthase-2 induction. Other diseases associated with Il -1,IL-6, IL-8 or TNF overproduction are set forth in WO 96/21654. P38γ MAPkinase (also known as ERK6 and stress activated protein kinase-3 orSAPK3) is a newly discovered member of the MAP kinase family. However,unlike the other P38 family members which are expressed in many tissues,P38γ is expressed at highest levels in skeletal muscle [Li et al.,Biochem Biophys Res Commun 228, 334-340 (1996); Enslen et al., J BiolChem 273, 1741-1748 (1998); Raingeaud et al., J. Biol. Chem. 270,7420-7426 (1995)]. Thus P38γ may have a unique function related tomuscle morphogenesis, and it may be a potential target for treatingdegenerative diseases occurring in muscle tissue.

[0005] Compounds that selectively inhibit P38γ and not P38α would behighly desirable. It would be useful to have new treatments for muscledegenerative diseases using compounds that do not suppress theinflammatory response or other functions of P38α. However, the design ofinhibitors that are selective for any particular MAP kinase, such asP38γ, is challenging due to the structural similarity of the MAPkinases. Therefore, it would be advantageous to have a detailedunderstanding of the structures of the various MAP kinases in order toexploit any subtle differences that may exist among them.

[0006] A general approach to designing inhibitors that are selective foran enzyme target is to determine how a putative inhibitor interacts withthe three dimensional structure of the enzyme. For this reason it isuseful to obtain the enzyme protein in crystal form and perform X-raydiffraction techniques to determine its three dimensional structurecoordinates. If the enzyme is crystallized as a complex with a ligand,one can determine both the shape of the enzyme binding pocket when boundto the ligand, as well as the amino acid residues that are capable ofclose contact with the ligand. By knowing the shape and amino acidresidues in the binding pocket, one may design new ligands that willinteract favorably with the enzyme. With such structural information,available computational methods may be used to predict how strong theligand binding interaction will be. Such methods thus enable the designof inhibitors that bind strongly, as well as selectively to the targetenzyme.

[0007] Crystal structures are known for some of the MAP kinases; forexample, unphosphorylated JNK3, unphosphorylated P38α, and ERK2 in bothphosphorylated and unphosphorylated forms. Phosphorylated ERK2 isreported to exist as a dimer in both solution and as a crystal. Theunphosphorylated forms of JNK3, ERK2 and P38α, on the other hand, arereported to be monomeric. [Tong et al., Nat Struct Biol 4, 311-316(1997); Wilson and Su, Chem Biol 4, 423-431 (1997); Xie et al.,Structure 6, 983-991 (1998); Zhang et al., Nature 367, 704-711 (1994);Canagarajah et al., Cell 90, 859-869 (1997); Wilson and Su, J Biol Chem271, 27696-27700 (1996)]

[0008] The crystal structure reported for P38α is based onunphosphorylated protein. However, it is the phosphorylated or activatedform of the enzyme that is able to phosphorylate its substrate enzyme.In order to disrupt the phosphorylation of the substrate, and producethe desired clinical effect, a small molecule inhibitor would likely actby blocking a phosphorylated form of P38. Thus, the most suitable targetfor drug design is the active or phosphorylated form. While thestructure of the unphosphorylated enzyme is often used for drug designpurposes, there is an inherent uncertainty as to whether thephosphorylated and unphosphorylated forms would bind a designedinhibitor with equal affinity.

[0009] A class of pyridinylimidazole compounds are known to inhibit P38αMAP kinase [Lee et al., Nature 372, 739-746 (1994)]. These inhibitorshave been shown to bind in the ATP binding site of P38α [Young et al., JBiol Chem 272, 12116-12121 (1997); Tong et al., Nat Struct Biol 4,311-316 (1997); Wilson et al., Chem Biol 4, 423-431 (1997)]. However,the pyridinylimidazoles reportedly do not inhibit the activity of ERK2,JNK3, or P38γ. This observed selectivity is interesting because theamino acid sequence in the ATP binding site of the various kinases areknown to be highly conserved [Fox et al., Protein Science 7, 2249-2255(1998); Xie et al., supra; Wilson and Su, supra; Enslen et al., J BiolChem 273, 1741-1748 (1998)].

[0010] As there is a need for compounds that selectively inhibit aparticular MAP kinase, it would be desirable to have improved methodsthat facilitate the design of such compounds. For this purpose,knowledge of the three dimensional structure coordinates of an activatedP38 protein would be useful. Such information would aid in identifyingand designing potential inhibitors of particular P38 proteins which, inturn, are expected to have therapeutic utility.

SUMMARY OF THE INVENTION

[0011] This invention provides certain crystallized, proteinkinase-ligand complexes, in particular P38-ligand complexes, and theirstructure coordinates. The structure coordinates are based on thestructure of a phosphorylated P38γ-ligand complex that has now beensolved and which reveals new structural information useful forunderstanding the activated states of other, related kinase proteins asdescribed herein. The key structural features of the proteins,particularly the shape of the substrate binding site, are useful inmethods for designing or identifying selective inhibitors of the proteinkinases, particularly P38, and in solving the structures of otherproteins with similar features.

[0012] The invention also provides a computer which which is programmedwith the structure coordinates of the activated P38 binding site. Such acomputer, appropriately programmed and attached to the necessary viewingdevice, is capable of displaying a three-dimensional graphicalrepresentation of a molecule or molecular complex comprising suchbinding sites or similarly shaped homologous binding pockets.

[0013] The invention also provides a method for determining at least aportion of the three-dimensional structure of other molecules ormolecular complexes which contain at least some features that arestructurally similar to P38γ, particularly P38α, P38β, P38δ and otherP38 isoforms. This is achieved by using at least some of the structuralcoordinates obtained for a phosphorylated P38 complex.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 lists the atomic structure coordinates for phosphorylatedP38γ in complex with MgAMP-PNP as derived by X-ray diffraction from acrystal of that complex. The following abbreviations are used in FIG. 1:

[0015] “Atom type” refers to the element whose coordinates are measured.The first letter in the column defines the element.

[0016] “X, Y, Z” crystallographically define the atomic position of theelement measured.

[0017] “B” is a thermal factor that measures movement of the atom aroundits atomic center.

[0018] “Occ” is an occupancy factor that refers to the fraction of themolecules in which each atom occupies the position specified by thecoordinates. A value of “1” indicates that each atom has the sameconformation, i.e., the same position, in all molecules of the crystal.

[0019]FIG. 1a is an overview of the phosphorylated P38γ.

[0020]FIG. 2 is a superimposition of unphosphorylated P38γ andphosphorylated P38γ.

[0021]FIG. 3 is a detailed stereo view of the activation loop.

[0022]FIG. 4 is a stereo view of the AMP-PNP bound in the active site.

[0023]FIG. 5 is a comparison of the active sites of activated P38γ withP38α (a) and cAPK or cyclic AMP dependent protein kinase (b).

[0024]FIG. 6 is a comparison of activated phosphorylation loops fromP38γ (dark orange), ERK2 (dark blue), and cAPK (red).

[0025]FIG. 7 shows a diagram of a system used to carry out theinstructions encoded by the storage medium of FIGS. 8 and 9.

[0026]FIG. 8 shows a cross section of a magnetic storage medium.

[0027]FIG. 9 shows a cross section of a optically-readable data storagemedium.

DETAILED DESCRIPTION OF THE INVENTION

[0028] This invention provides certain crystallized, proteinkinase-ligand complexes, in particular P38-ligand complexes, and theirstructure coordinates. The structure coordinates are based on thestructure of a phosphorylated P38γ complex that has now been solved andwhich reveals new structural information regarding the activated statesof other, related kinase proteins as described herein. The keystructural features of the protein, particularly the shape of thesubstrate binding site, are useful in methods for designing inhibitorsof the P38 and in solving the structures of other proteins with similarfeatures.

[0029] In describing protein structure and function, reference is madeto amino acids comprising the protein. The amino acids may also bereferred to by their conventional abbreviations, as shown in the tablebelow. A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys =Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = Ile = Isoleucine N =Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine F = Phe =Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu =Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly = Glycine R= Arg = Arginine S = Ser = Serine H = His = Histidine

[0030] This invention also provides a crystallizable composition fromwhich the crystallized protein is obtained. The crystallizablecomposition preferably comprises a phosphorylated P38 protein complexedwith a substrate or ligand. The ligand may be any ligand capable ofbinding to the P38 protein, and is preferably a ligand that binds to theATP binding site of the protein. Examples of such ligands are smallmolecule inhibitors of the particular P38 as well as non-hydrolyzableATP analogs and suicide substrates. Non-hydrolyzable ATP analogs usefulin the crystallizable compositions of this invention include AMP-PCH₂P,AMP-PSP and AMP-PNP where the oxygen linking the second and thirdphosphates of the ATP analogs is replaced by CH₂, S and NH,respectively. An example of a suicidal substrate is 5′-(p-fluorosulfonylbenzoyl)adenosine (FSBA). Preferably, the crystallizable compositions ofthis invention comprise AMP-PNP as the substrate. It is preferred thatthe composition further comprise divalent cations, especially magnesiumor magnanese cations, which may be introduced in any suitable manner.For example, the cations may be introduced by incubating the desiredligand with a suitable metal salt such as MgCl₂ prior to incubation withthe phosphorylated P38 protein.

[0031] It has been found that the crystallization of the phosphorylatedP38 protein is sensitive to buffer conditions. Thus, in a preferredembodiment, the crystallizable compositions of this invention furthercomprise a suitable glycol such as ethylene glycol, polyethylene glycol(PEG), PEG-monomethyl ether or mixtures thereof, preferably PEG 4000, asan aqueous solution containing between about 10 to 35% of the glycol byvolume of solution, a salt, such as sodium acetate at about 50 to 200mM, a reducing agent, such as dithiothreitol (DTT) at between about 1 to10 mM, a detergent such as C12E9 at about 0.01 to 0.05%, and a bufferthat maintains pH at between about 8.0 and 9.0. An example of a suitablebuffer is 100 mM Tris at pH 8.5.

[0032] By applying standard crystallization protocols to the abovedescribed crystallizable compositions, crystals of the phosphorylatedP38 protein complex may be obtained. Thus, one aspect of this inventionrelates to a method of preparing phosphorylated P38-containing crystals.The method comprises the steps of

[0033] (a) obtaining a crystallizable composition comprising aphosphorylated P38 protein, divalent cations, and a ligand capable ofbinding to the protein, and

[0034] (b) subjecting the composition of step (a) to conditions whichpromote crystallization.

[0035]FIG. 1 shows the structure coordinates of a phosphorylated P38γprotein complexed with MgAMP-PNP. The manner of obtaining thesestructure coordinates, interpretation of the coordinates and theirutility in understanding the protein structure, as described herein,will be understood by those of skill in the art and by reference tostandard texts such as Crystal Structure Analysis, Jenny PickworthGlusker and Kenneth N. Trueblood, 2nd Ed. Oxford University Press, 1985,New York; and Principles of Protein Structure, G. E. Schulz and R. H.Schirmer, Springer-Verlag, 1985, New York.

[0036] Those of skill in the art understand that a set of structurecoordinates for an enzyme or an enzyme-complex or a portion thereof, isa relative set of points that define a shape in three dimensions. Thus,it is possible that an entirely different set of coordinates coulddefine a similar or identical shape. Moreover, slight variations in theindividual coordinates will have little effect on overall shape. Interms of binding pockets, these variations would not be expected tosignificantly alter the nature of ligands that could associate withthose pockets.

[0037] These variations in coordinates may be generated because ofmathematical manipulations of the P38γ/MgAMP-PNP structure coordinates.For example, the structure coordinates set forth in FIG. 1 could bemanipulated by crystallographic permutations of the structurecoordinates, fractionalization of the structure coordinates, integeradditions or subtractions to sets of the structure coordinates,inversion of the structure coordinates or any combination of the above.

[0038] Alternatively, modifications in the crystal structure due tomutations, additions, substitutions, and/or deletions of amino acids, orother changes in any of the components that make up the crystal couldalso account for variations in structure coordinates. If such variationsare within an acceptable standard error as compared to the originalcoordinates, the resulting three-dimensional shape is considered to bethe same. Thus, for example, a ligand that bound to the active sitebinding pocket of P38γ would also be expected to bind to another bindingpocket whose structure coordinates defined a shape that fell within theacceptable error.

[0039] The term “binding pocket” refers to a region of the protein that,as a result of its shape, favorably associates with a ligand orsubstrate. The term “P38γ-like binding pocket” refers to a portion of amolecule or molecular complex whose shape is sufficiently similar to theP38γ binding pockets as to bind common ligands. This commonality ofshape may be quantitatively defined by a root mean square deviation(rmsd) from the structure coordinates of the backbone atoms of the aminoacids that make up the binding pockets in P38γ (as set forth in FIG. 1).The method of performing this rmsd calculation is described below.

[0040] The “active site binding pockets” or “active site” of P38γ refersto the area on the P38γ enzyme surface where the substrate binds. Inresolving the crystal structure of phosphorylated P38γ in complex withMgAMP-PNP, applicants have determined that P38γ amino acids Val33,Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112,Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160,Leu170, Asp171, Gly173, and Leu174 are within 5 Å of and therefore closeenough to interact with MgAMP-PNP. These amino acids are hereinafterreferred to as the “SET 5A amino acids.” Thus, a binding pocket definedby the structural coordinates of those amino acids, as set forth in FIG.1; or a binding pocket whose root mean square deviation from thestructure coordinates of the backbone atoms of those amino acids of notmore than about 1.15 angstroms (Å) is considered a P38γ-like bindingpocket of this invention.

[0041] Applicants have also determined that in addition to the P38γamino acids set forth above, Pro32, Cys42, Ser43, Val53, Ile55, Lys57,Leu58, Thr59, Arg70, Glu74, Gly88, Leu107, Val108, Leu116, Gly117,Pro156, Leu159, Val161, Lys168, Phe172, Ala175, and Thr188 are within 8Å of bound MgAMP-PNP and therefore are also close enough to interactwith that substrate. These amino acids, in addition to the SET 5A aminoacids, are hereinafter referred to as the “SET 8A amino acids.” Thus, ina preferred embodiment, a binding pocket defined by the structuralcoordinates of the amino acids within 8 Å of bound MgAMP-PNP, as setforth in FIG. 1; or a binding pocket whose root mean square deviationfrom the structure coordinates of the backbone atoms of those aminoacids of not more than about 1.15 Å is considered a preferred P38γ-likebinding pocket of this invention.

[0042] It will be readily apparent to those of skill in the art that thenumbering of amino acids in other isoforms of P38 may be different thanthat set forth for P38γ. Corresponding amino acids in other isoforms ofP38 are easily identified by visual inspection of the amino acidsequences or by using commercially available homology software programs,as further described below.

[0043] Various computational analyses may be used to determine whether aprotein or the binding pocket portion thereof is sufficiently similar tothe P38γ binding pockets described above. Such analyses may be carriedout in well known software applications, such as the MolecularSimilarity application of QUANTA (Molecular Simulations Inc., San Diego,Calif.) version 4.1, and as described in the accompanying User's Guide.

[0044] For the purpose of this invention, a rigid fitting method wasconveniently used to compare protein structures. Any molecule ormolecular complex or binding pocket thereof having a root mean squaredeviation of conserved residue backbone atoms (N, Cα, C, O) of less thanabout 1.15 Å when superimposed on the relevant backbone atoms describedby structure coordinates listed in FIG. 1 are considered identical. Morepreferably, the root mean square deviation is less than about 1.0 Å.

[0045] The P38 X-ray coordinate data, when used in conjunction with acomputer programmed with software to translate those coordinates intothe 3-dimensional structure of p38γ may be used for a variety ofpurposes, especially for purposes relating to drug discovery. Suchsoftware for generating three-dimensional graphical representations areknown and commercially available. The ready use of the coordinate datarequires that it be stored in a computer-readable format. Thus, inaccordance with the present invention, data capable of being displayedas the three dimensional structure of P38γ and portions thereof andtheir structurally similar homologues is stored in a machine-readablestorage medium, which is capable of displaying a graphicalthree-dimensional representation of the structure.

[0046] Therefore, another embodiment of this invention provides amachine-readable data storage medium, comprising a data storage materialencoded with machine readable data which, when used by a machineprogrammed with instructions for using said data, displays a graphicalthree-dimensional representation of a molecule or molecular complexcomprising a binding pocket defined by structure coordinates of the P38γSET 5A amino acids, or preferably the P38γ SET 8A amino acids, or ahomologue of said molecule or molecular complex, wherein said homologuecomprises a binding pocket that has a root mean square deviation fromthe backbone atoms of said amino acids of not more than about 1.15 Å.

[0047] Even more preferred is a machine-readable data storage mediumthat is capable of displaying a graphical three-dimensionalrepresentation of a molecule or molecular complex that is defined by thestructure coordinates of all of the amino acids in FIG. 1 or a homologueof said molecule or molecular complex, wherein said homologue has a rootmean square deviation from the backbone atoms of all of the amino acidsin FIG. 1 of not more than about 1.15 Å.

[0048] According to an alternate embodiment, the machine-readable datastorage medium comprises a data storage material encoded with a firstset of machine readable data which comprises the Fourier transform ofthe structure coordinates set forth in FIG. 1, and which, when using amachine programmed with instructions for using said data, can becombined with a second set of machine readable data comprising the X-raydiffraction pattern of another molecule or molecular complex todetermine at least a portion of the structure coordinates correspondingto the second set of machine readable data.

[0049] For example, the Fourier transform of the structure coordinatesset forth in FIG. 1 may be used to determine at least a portion of thestructure coordinates of other P38s, such as P38β, and P38δ and isoformsof P38β, P38δ or P38γ. The structure coordinates in FIG. 1 and theFourier transform of the coordinates are especially useful fordetermining the coordinates of other P38s in phosphorylated form.

[0050] According to an alternate embodiment, this invention provides acomputer for producing a three-dimensional representation of a moleculeor molecular complex, wherein said molecule or molecular complexcomprises a binding pocket defined by the P38γ SET 5A amino acids, orpreferably the P38γ SET 8A amino acids, or a homologue of said moleculeor molecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than 1.15 Å, wherein said computer comprises:

[0051] (a) a machine readable data storage medium comprising a datastorage material encoded with machine-readable data, wherein saidmachine readable data comprises the structure coordinates of P38γ orportions thereof;

[0052] (b) a working memory for storing instructions for processing saidmachine-readable data;

[0053] (c) a central-processing unit coupled to said working memory andto said machine-readable data storage medium, for processing saidmachine-readable data into said three-dimensional representation; and

[0054] (d) an output hardware coupled to said central processing unit,for receiving said three Dimensional representation.

[0055]FIG. 7 demonstrates one version of these embodiments. System 10includes a computer 11 comprising a central processing unit (“CPU”) 20,a working memory 22 which may be, eg., RAM (random-access memory) or“core” memory, mass storage memory 24 (such as one or more disk drivesor CD-ROM drives), one or more cathode-ray tube (“CRT”) displayterminals 26, one or more keyboards 28, one or more input lines 30, andone or more output lines 40, all of which are interconnected by aconventional bi-directional system bus 50.

[0056] Input hardware 36, coupled to computer 11 by input lines 30, maybe implemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems 32 connectedby a telephone line or dedicated data line 34. Alternatively oradditionally, the-input hardware 36 may comprise CD-ROM drives or diskdrives 24. In conjunction with display terminal 26, keyboard 28 may alsobe used as an input device.

[0057] Output hardware 46, coupled to computer 11 by output lines 40,may similarly be implemented by conventional devices. By way of example,output hardware 46 may include CRT display terminal 26 for displaying agraphical representation of a binding pocket of this invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer 42, so that hard copy output may be produced, or adisk drive 24, to store system output for later use.

[0058] In operation, CPU 20 coordinates the use of the various input andoutput devices 36, 46 coordinates data accesses from mass storage 24 andaccesses to and from working memory 22, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system 10 areincluded as appropriate throughout the following description of the datastorage medium.

[0059]FIG. 8 shows a cross section of a magnetic data storage medium 100which can be encoded with a machine-readable data that can be carriedout by a system such as system 10 of FIG. 7. Medium 100 can be aconventional floppy diskette or hard disk, having a suitable substrate101, which may be conventional, and a suitable coating 102, which may beconventional, on one or both sides, containing magnetic domains (notvisible) whose polarity or orientation can be altered magnetically.Medium 100 may also have an opening (not shown) for receiving thespindle of a disk drive or other data storage device 24. The magneticdomains of coating 102 of medium 100 are polarized or oriented so as toencode in manner which may be conventional, machine readable data suchas that described herein, for execution by a system such as system 10 ofFIG. 7.

[0060]FIG. 9 shows a cross section of an optically-readable data storagemedium 110 which also can be encoded with such a machine-readable data,or set of instructions, which can be carried out by a system such assystem 10 of FIG. 7. Medium 110 can be a conventional compact disk readonly memory (CD-ROM) or a rewritable medium such as a magneto-opticaldisk which is optically readable and magneto-optically writable. Medium100 preferably has a suitable substrate 111, which may be conventional,and a suitable coating 112, which may be conventional, usually of oneside of substrate 111.

[0061] In the case of CD-ROM, as is well known, coating 112 isreflective and is impressed with a plurality of pits 113 to encode themachine-readable data. The arrangement of pits is read by reflectinglaser light off the surface of coating 112. A protective coating 114,which preferably is substantially transparent, is provided on top ofcoating 112.

[0062] In the case of a magneto-optical disk, as is well known, coating112 has no pits 113, but has a plurality of magnetic domains whosepolarity or orientation can be changed magnetically when heated above acertain temperature, as by a laser (not shown). The orientation of thedomains can be read by measuring the polarization of laser lightreflected from coating 112. The arrangement of the domains encodes thedata as described above.

[0063] As mentioned above, the P38γ X-ray coordinate data is useful forscreening and identifying drugs that inhibit P38, especiallyphosphorylated P38. For example, the structure encoded by the data maybe computationally evaluated for its ability to associate with putativesubstrates or ligands. Such compounds that associate with p38γ mayinhibit p38γ, and are potential drug candidates. Additionally oralternatively, the structure encoded by the data may be displayed in agraphical three-dimensional representation on a computer screen. Thisallows visual inspection of the structure, as well as visual inspectionof the structure's association with the compounds.

[0064] Thus, according to another embodiment, this invention relates toa method for evaluating the potential of a compound to associate with amolecule or molecular complex comprising a binding pocket defined by thestructure coordinates of the P38γ SET 5A amino acids, or preferably theP38γ SET 8A amino acids, or a homologues of said molecule or molecularcomplex, wherein said homologue comprises a binding pocket that has aroot mean square deviation from the backbone atoms of said amino acidsof not more than about 1.15 Å.

[0065] This method comprises the steps of:

[0066] a) creating a computer model of the binding pocket usingstructure coordinates wherein the root mean square deviation betweensaid structure coordinates and the structure coordinates of the P38γamino acids Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110,Phe111, Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157,Asn158, Ala160, Leu170, Asp171, Gly173, and Leu174 according to FIG. 1is not more than about 1.15 Å;

[0067] b) employing computational means to perform a fitting operationbetween the chemical entity and said computer model of the bindingpocket; and

[0068] c) analyzing the results of said fitting operation to quantifythe association between the chemical entity and the binding pocketmodel.

[0069] The term “chemical entity”, as used herein, refers to chemicalcompounds or ligands, complexes of at least two chemical compounds, andfragments of such compounds or complexes.

[0070] Even more preferably, the method evaluates the potential of achemical entity to associate with a molecule or molecular complexdefined by the structure coordinates of all of the P38γ amino acids, asset forth in FIG. 1, or a homologue of said molecule or molecularcomplex having a root mean square deviation from the backbone atoms ofsaid amino acids of not more than 1.15 Å.

[0071] Alternatively, the structural coordinates of the P38γ bindingpocket can be utilized in a method for identifying a potential agonistor antagonist of a molecule comprising a P38γ-like binding pocket. Thismethod comprises the steps of:

[0072] (a) using atomic coordinates of the P38γ SET 5A amino acids±aroot mean square deviation from the backbone atoms of said amino acidsof not more than about 1.15 Å, to generate a three-dimensional structureof molecule comprising a P38γ-like binding pocket;

[0073] (b) employing said three-dimensional structure to design orselect said potential agonist or antagonist;

[0074] (c) synthesizing said agonist or antagonist; and

[0075] (d) contacting said agonist or antagonist with said molecule todetermine the ability of said potential agonist or antagonist tointeract with said molecule.

[0076] More preferred is the use of the atomic coordinates of the P38γSET 8A amino acids, ±a root mean square deviation from the backboneatoms of said amino acids of not more than 1.15 Å, to generate athree-dimensional structure of molecule comprising a p38γ-like bindingpocket. Most preferred is when the atomic coordinates of all the aminoacids of P38γ according to FIG. 1±a root mean square deviation from thebackbone atoms of said amino acids of not more than 1.15 Å, are used togenerate a three-dimensional structure of molecule comprising aP38γ-like binding pocket.

[0077] For the first time, the present invention permits the use ofmolecular design techniques to identify, select or design potentialinhibitors of p38, based on the structure of a phosphorylated p38γ-likebinding pocket. Such a predictive model is valuable in light of the highcosts associated with the preparation and testing of the many diversecompounds that may possibly bind to the p38 protein.

[0078] According to this invention, a potential p38 inhibitor may now beevaluated for its ability to bind a P38γ-like binding pocket prior toits actual synthesis and testing. If a proposed compound is predicted tohave insufficient interaction or association with the binding pocket,preparation and testing of the compound is obviated. However, if thecomputer modeling indicates a strong interaction, the compound may thenbe obtained and tested for its ability to bind. Testing to confirmbinding may be performed using assays such as described in Example 6.

[0079] A potential inhibitor of a P38γ-like binding pocket may becomputationally evaluated by means of a series of steps in whichchemical entities or fragments are screened and selected for theirability to associate with the P38γ-like binding pockets.

[0080] One skilled in the art may use one of several methods to screenchemical entities or fragments for their ability to associate with aP38γ-like binding pocket. This process may begin by visual inspectionof, for example, a P38γ-like binding pocket on the computer screen basedon the P38γ structure coordinates in FIG. 1 or other coordinates whichdefine a similar shape generated from the machine-readable storagemedium. Selected fragments or chemical entities may then be positionedin a variety of orientations, or docked, within that binding pocket asdefined above. Docking may be accomplished using software such as Quantaand Sybyl, followed by energy minimization and molecular dynamics withstandard molecular mechanics force fields, such as CHARMM and AMBER.

[0081] Specialized computer programs may also assist in the process ofselecting fragments or chemical entities. These include:

[0082] 1. GRID (P. J. Goodford, “A Computational Procedure forDetermining Energetically Favorable Binding Sites on BiologicallyImportant Macromolecules”, J. Med. Chem., 28, pp. 849-857 (1985)). GRIDis available from Oxford University, Oxford, UK.

[0083] 2. MCSS (A. Miranker et al., “Functionality Maps of BindingSites: A Multiple Copy Simultaneous Search Method.” Proteins,:Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS isavailable from Molecular Simulations, San Diego, Calif.

[0084] 3. AUTODOCK (D. S. Goodsell et al., “Automated Docking ofSubstrates to Proteins by Simulated Annealing”, Proteins: Structure,Function, and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is availablefrom Scripps Research Institute, La Jolla, Calif.

[0085] 4. DOCK (I. D. Kuntz et al., “A Geometric Approach toMacromolecule-Ligand Interactions”, J. Mol. Biol., 161, pp. 269-288(1982)). DOCK is available from University of California, San Francisco,Calif.

[0086] Once suitable chemical entities or fragments have been selected,they can be designed or assembled into a single compound or complex.Assembly may be preceded by visual inspection of the relationship of thefragments to each other on the three-dimensional image displayed on acomputer screen in relation to the structure coordinates of P38γ. Thiswould be followed by manual model building using software such as Quantaor Sybyl [Tripos Associates, St. Louis, Mo.].

[0087] Useful programs to aid one of skill in the art in connecting theindividual chemical entities or fragments include:

[0088] 1. CAVEAT (P. A. Bartlett et al., “CAVEAT: A Program toFacilitate the Structure-Derived Design of Biologically ActiveMolecules”, in Molecular Recognition in Chemical and BiologicalProblems”, Special Pub., Royal Chem. Soc., 78, pp. 182-196 (1989); G.Lauri and P. A. Bartlett, “CAVEAT: a Program to Facilitate the Design ofOrganic Molecules”, J. Comput. Aided Mol. Des. , 8, pp. 51-66 (1994)).CAVEAT is available from the University of California, Berkeley, Calif.

[0089] 2. 3D Database systems such as ISIS (MDL Information Systems, SanLeandro, Calif.). This area is reviewed in Y. C. Martin, “3D DatabaseSearching in Drug Design”, J. Med. Chem., 35, pp. 2145-2154 (1992).

[0090] 3. HOOK (M. B. Eisen et al, “HOOK: A Program for Finding NovelMolecular Architectures that Satisfy the Chemical and StericRequirements of a Macromolecule Binding Site”, Proteins: Struct.,Funct., Genet., 19, pp. 199-221 (1994). HOOK is available from MolecularSimulations, San Diego, Calif.

[0091] Instead of proceeding to build an inhibitor of a P38γ-likebinding pocket in a step-wise fashion one fragment or chemical entity ata time as described above, inhibitory or other P38γ binding compoundsmay be designed as a whole or “de novo” using either an empty bindingsite or optionally including some portion(s) of a known inhibitor(s).There are many de novo ligand design methods including:

[0092] 1. LUDI (H.-J. Bohm, “The Computer Program LUDI: A New Method forthe De Novo Design of Enzyme Inhibitors”, J. Comp. Aid. Molec. Design,6, pp. 61-78 (1992)). LUDI is available from Molecular SimulationsIncorporated, San Diego, Calif.

[0093] 2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)).LEGEND is available from Molecular Simulations Incorporated, San Diego,Calif.

[0094] 3. LeapFrog (available from Tripos Associates, St. Louis, Mo.).

[0095] 4. SPROUT (V. Gillet et al, “SPROUT: A Program for StructureGeneration)”, J. Comput. Aided Mol. Design, 7, pp. 127-153 (1993)).SPROUT is available from the University of Leeds, UK.

[0096] Other molecular modeling techniques may also be employed inaccordance with this invention [see, e.g., Cohen et al., “MolecularModeling Software and Methods for Medicinal Chemistry, J. Med. Chem.,33, pp. 883-894 (1990); see also, M. A. Navia and M. A. Murcko, “The Useof Structural Information in Drug Design”, Current Opinions inStructural Biology, 2, pp. 202-210 (1992); L. M. Balbes et al., “APerspective of Modern Methods in Computer-Aided Drug Design”, in Reviewsin Computational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd,Eds., VCH, New York, pp. 337-380 (1994); see also, W. C. Guida,“Software For Structure-Based Drug Design”, Curr. Opin. Struct. Biology,4, pp. 777-781 (1994)].

[0097] Once a compound has been designed or selected by the abovemethods, the efficiency with which-that entity may bind to a P38γbinding pocket may be tested and optimized by computational evaluation.For example, an effective P38γ binding pocket inhibitor must preferablydemonstrate a relatively small difference in energy between its boundand free states (i.e., a small deformation energy of binding). Thus, themost efficient P38γ binding pocket inhibitors should preferably bedesigned with a deformation energy of binding of not greater than about10 kcal/mole, more preferably, not greater than 7 kcal/mole. P38γbinding pocket inhibitors may interact with the binding pocket in morethan one of multiple conformations that are similar in overall bindingenergy. In those cases, the deformation energy of binding is taken to bethe difference between the energy of the free entity and the averageenergy of the conformations observed when the inhibitor binds to theprotein.

[0098] An entity designed or selected as binding to a P38γ bindingpocket may be further computationally optimized so that in its boundstate it would preferably lack repulsive electrostatic interaction withthe target enzyme and with the surrounding water molecules. Suchnon-complementary electrostatic interactions include repulsivecharge-charge, dipole-dipole and charge-dipole interactions.

[0099] Specific computer software is available in the art to evaluatecompound deformation energy and electrostatic interactions. Examples ofprograms designed for such uses include: Gaussian 94, revision C (M. J.Frisch, Gaussian, Inc., Pittsburgh, Pa. ©1995); AMBER, version 4.1 (P.A. Kollman, University of California at San Francisco, ©1995);QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. ©1995);Insight II/Discover (Molecular Simulations, Inc., San Diego, Calif.©1995); DelPhi (Molecular Simulations, Inc., San Diego, Calif. (©1995);and AMSOL (Quantum Chemistry Program Exchange, Indiana University).These programs may be implemented, for instance, using a SiliconGraphics workstation such as an Indigo² with “IMPACT” graphics. Otherhardware systems and software packages will be known to those skilled inthe art.

[0100] Another approach enabled by this invention, is the computationalscreening of small molecule databases for chemical entities or compoundsthat can bind in whole, or in part, to a P38γ binding pocket. In thisscreening, the quality of fit of such entities to the binding site maybe judged either by shape complementarity or by estimated interactionenergy [E. C. Meng et al., J. Comp. Chem., 13, 505-524 (1992)].

[0101] According to another embodiment, the invention provides compoundswhich associate with a P38γ-like binding pocket produced or identifiedby the method set forth above.

[0102] The structure coordinates set forth in FIG. 1 can also be used toaid in obtaining structural information about another crystallizedmolecule or molecular complex. This may be achieved by any of a numberof well-known techniques, including molecular replacement.

[0103] Therefore, in another embodiment this invention provides a methodof utilizing molecular replacement to obtain structural informationabout a molecule or molecular complex whose structure is unknowncomprising the steps of:

[0104] a) crystallizing said molecule or molecular complex of unknownstructure;

[0105] b) generating an X-ray diffraction pattern from said crystallizedmolecule or molecular complex; and

[0106] c) applying at least a portion of the structure coordinates setforth in FIG. 1 to the X-ray diffraction pattern to generate athree-dimensional electron density map of the molecule or molecularcomplex whose structure is unknown.

[0107] By using molecular replacement, all or part of the structurecoordinates of the P38γ/MgAMP-PNP complex as provided by this invention(and set forth in FIG. 1) can be used to determine the structure ofanother crystallized molecule or molecular complex more quickly andefficiently than attempting an ab initio structure determination.

[0108] Molecular replacement provides an accurate estimation of thephases for an unknown structures Phases are a factor in equations usedto solve crystal structures that can not be determined directly.Obtaining accurate values for the phases, by methods other thanmolecular replacement, is a time-consuming process that involvesiterative cycles of approximations and refinements and greatly hindersthe solution of crystal structures. However, when the crystal structureof a protein containing at least a homologous portion has been solved,the phases from the known structure provide a satisfactory estimate ofthe phases for the unknown structure.

[0109] Thus, this method involves generating a preliminary model of amolecule or molecular complex whose structure coordinates are unknown.bv orienting and positioning the relevant portion of the P38γ/MgAMP-PNPcomplex according to FIG. 1 within the unit cell of the crystal of theunknown molecule or molecular complex so as best to account for theobserved X-ray diffraction pattern of the crystal of the molecule ormolecular complex whose structure is unknown. Phases can then becalculated from this model and combined with the observed X-raydiffraction pattern amplitudes to generate an electron density map ofthe structure whose coordinates are unknown. This, in turn, can besubjected to any well-known model building and structure refinementtechniques to provide a final, accurate structure of the unknowncrystallized molecule or molecular complex [E. Lattman, “Use of theRotation and Translation Functions”, in Meth. Enzymol., 115, pp. 55-77(1985); M. G. Rossmann, ed., “The Molecular Replacement Method”, Int.Sci. Rev. Ser., No. 13, Gordon & Breach, New York (1972)].

[0110] The structure of any portion of any crystallized molecule ormolecular complex that is sufficiently homologous to any portion of theP38γ/MgAMP-PNP complex can be resolved by this method.

[0111] In a preferred embodiment, the method of molecular replacement isutilized to obtain structural information about another P38, such asP38α, P38β, P38δ, or isoforms of P38β, P38δ or P38γ. The structurecoordinates of P38γ as provided by this invention are particularlyuseful in solving the structure of other isoforms of P38γ or P38γcomplexes.

[0112] Furthermore, the structure coordinates of P38γ as provided bythis invention are useful in solving the structure of P38γ proteins thathave amino acid substitutions, additions and/or deletions (referred tocollectively as “P38γ mutants”, as compared to naturally occurring P38γisoforms). These P38γ mutants may optionally be crystallized inco-complex with a chemical entity, such as a non-hydrolyzable ATPanalogue or a suicide substrate. The crystal structures of a series ofsuch complexes may then be solved by molecular replacement and comparedwith that of wild-type p38γ. Potential sites for modification within thevarious binding sites of the enzyme may thus be identified. Thisinformation provides an additional tool for determining the mostefficient binding interactions such as, for example, increasedhydrophobic interactions, between P38γ and a chemical entity orcompound.

[0113] All of the complexes referred to above may be studied usingwell-known X-ray diffraction techniques and may be refined versus 1.5-3Aresolution X-ray data to an R value of about 0.22 or less using computersoftware, such as X-PLOR [Yale University, ©1992, distributed byMolecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth.Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press(1985)]. This information may thus be used to optimize known P38γinhibitors, and more importantly, to design new P38γ inhibitors.

[0114] The structure coordinates described above may also be used toderive the dihedral angles, φ and ψ, that define the conformation of theamino acids in the protein backbone. As will be understood by thoseskilled in the art, the φ_(n) angle refers to the rotation around thebond between the alpha carbon and the nitrogen, and the ψ_(n) anglerefers to the rotation around the bond between the carbonyl carbon andthe alpha carbon. The subscript “n” identifies the amino acid whoseconformation is being described [for a general reference, see Blundelland Johnson, Protein Crystallography, Academic Press, London, 1976].

[0115] Surprisingly, it has now been found that for the crystallineP38γ-ligand complex, the conformation of Gly113 is very different fromthe conformations reported for corresponding amino acids in otherprotein kinases. In order to compare the conformations of P38γ and otherprotein kinases at a particular amino acid site, such as Gly113, alongthe polypeptide backbone well-known procedures may be used for doingsequence alignments of the amino acids. Such sequence alignments allowfor the equivalent or corresponding sites to be compared. One suchmethod for doing a sequence alignment is the “bestfit” program availablefrom Genetics Computer Group which uses the local homology algorithmdescribed by Smith and Waterman in Advances in Applied Mathematics 2;482 (1981).

[0116] A suitable amino acid sequence alignment will require that theproteins being aligned share a minimum percentage of identical aminoacids. Generally, a first protein being aligned with a second proteinshould share in excess of about 35% identical amino acids with thesecond protein. Hanks et al., Science, 241, 42 (1988); Hanks and Quinn,Methods in Enzymology, 200, 38 (1991).

[0117] Equivalents of the Gly113 residue of p38γ may also be identifiedby its functional position. Gly113 is the amino acid residue thatimmediately follows sequentially the amino acid residue that donates, oris capable of donating, a hydrogen bond to the N1 nitrogen of theadenosine ring of ATP or an ATP analog, if such ATP or ATP analog wereto be in the binding pocket comprising the Gly113 residue. The abilityof the amino acid to donate such a hydrogen bond occurs as the result ofthe spatial position of the amino acid in the binding pocket or theprotein. As used herein, the term “corresponding amino acid” or“equivalent amino acid” refers to a particular amino acid in a proteinkinase that corresponds to another, particular amino acid in a differentprotein kinase as determined by sequence alignment and/or its functionalposition.

[0118] Table 1 shows the sequence alignments for selected proteinkinases where corresponding amino acids are shown in the same column.The amino acid numbering is based on the assignments given in theSwiss-Prot database which is an international protein sequence databasedistributed by the European Bioinformatics Institute (EBI) in Geneva,Switzerland. The database can be found at www.ebi.ac.uk/swissprot.Erk6_HUMAN is the database protein name for P38γ. The ten amino acidsimmediately preceding G113 of P38γ are given starting with T103. Thus,for example, Gly113 of P38γ corresponds or is equivalent to thefollowing: Gly110 of P38α (MP38_HUMAN), Glu107 of mouse ERK2, and Asp150of human JNK3. The last column of Table 1 shows the Swiss-Prot databaseaccession number. TABLE 1 Sequence Alignments for Selected ProteinsCorresponding Amino Acid Sequences Using Swiss-Prot Access Protein AminoAcid Numbering Number ERK6_HUMAN T103 D F Y L V M P F M112 G113 P53778MP38_HUMAN N100 D V Y L V T H L M109 G110 Q16539 ERK2_HUMAN K99 D V Y IV Q D L M108 E109 P28482 ERK2_MOUSE K97 D V Y I V Q D L M106 E107 P27703JNK3_HUMAN Q140 D V Y L V M E L M149 D150 P53779 KAPA_MOUSE S114 N L Y MV M E Y V123 A124 P05132 INSR_HUMAN Q1097 P T L V V M E L M1106 A1107P06213 LCK_HUMAN E309 P I Y I I T E Y M318 E319 P06239 ZA70_HUMAN E408 AL M L V M E M A417 G418 P43403 PKD1_DICDI T107 K I H F I M E Y A116 G117P34100 KPCI_YEAST N898 R I Y F A M E F I907 G908 P24583 CLK1_HUMAN G235H I C I V F E L L244 G245 P49759 CLK2_HUMAN G237 H M C I S F E L L246G247 P49760 DOA_DROME G243 H M C I V F E M L252 G253 P49762 DSK1_SCHPOA160 H V C M V F E V L169 G170 P36616 MKK1_YEAST S293 S I Y I A M E YM302 G303 P32490 MKK2_YEAST S286 S I Y I A M E Y M295 G296 P32491NIMA_EMENI Q83 D L Y L Y M E Y C92 G93 P11837 KMOS_HUMAN S133 L G T I IM E F G142 G143 P00540 KC1A_HUMAN D84 Y N V L V M D L L93 G94 P48729KC1B_BOVIN D84 Y N V L V M D L L93 G94 P35507 KC1D_HUMAN D76 Y N V M V ME L L85 G86 P48730 CK11_YEAST L136 H N I L V I D L L145 G146 P23291CK12_YEAST L143 H N I L V I D L L152 G153 P23292 HR25_YEAST 576 Y N A MV I D L L85 G86 P29295 KNS1_YEAST N387 H I C L V T D L Y396 G397 P32350KYK1_DICDI D1360 H H C I V T E W M1369 G1370 P18160 CKI1_SCHPO L79 H N VL V I D L L88 G89 P40233 CDK2_HUMAN N74 K L Y L V F E F L83 H84 P24941KPBG_HUMAN T97 F F F L V F D L M106 K107 Q16816 KCC1_HUMAN G89 H L Y L IM Q L V98 S99 Q14012

[0119] As noted above, the conformation of Gly113 is very different fromthe conformations reported for corresponding amino acids in otherprotein kinases. For Gly113 of the P38γ-AMPPNP complex, ψ₁₁₂ was foundto be about 24 degrees and φ₁₁₃ was found to be about 96 degrees. Table2 shows the dihedral angles for Met112 and Gly113 of P38γ-AMPPNP complexand how these angles compare to those of the corresponding amino acidsin other MAP kinases whose crystal structures have been reported. Theprotein names for the known proteins are provided as their Protein DataBank™ (pdb) accession numbers. The Protein Data Bank is an internationalrepository for three dimensional structures and can be located atwww.rcsb.org/pdb/. TABLE 2 Dihedral Angles (in degrees) for Met112 andGly113 and Equivalents in P38_ and Other Protein Kinases Met 112 Gly 113Protein φ ψ φ ψ P38γ-AMPPNP −106.2 23.8 96.24 −90.6 P38α-ligand^(a)−80.8 −26.5 95.7 −22.5 1ERK^(b) −119.1 131.7 −51.6 −55.6 2ERK^(c) −99.5130.3 −42.7 −49.9 1p38^(d) −92.7 128.4 −82.1 −103.2 1ATP^(e) −96.6 89.1−56.1 −30.1 1JNK^(f) −105.3 170.6 −92.2 −22.8 1IR3^(g) −112.7 87.9 −44.2−38.4 1IRK^(h) −85.6 109.9 −40.7 −38.4 3LCK^(i) −121.7 105.9 −53.3 −38.2

[0120] It is well-recognized that there will be some variability in theconformations of corresponding amino acids in similar or identicalproteins when the protein crystallization and structure determinationare repeated. This variability in the φ and ψ dihedral angles may beapproximated by reference to Ramachandran plots comparing theconformations obtained for two or more identical or similar proteins[Blundell and Johnson, Protein Crystallography, Academic Press, London,1976]. It may be expected that the dihedral angles of equivalent aminoacid residues in identical or similar proteins will vary as much asabout 45° or more.

[0121] It should be noted that the amino acid numbering defined in theProtein Data Banks may be offset from the numbering given in theSwiss-Prot database. This offset, when it occurs, will be readilyunderstood by those skilled in the art. Thus, the sequences of thoseproteins that are listed in both databases may be easily compareddespite offsets in amino acid numbering that may occur. Examples of suchoffsets occur for INSR_HUMAN where A1107 according to Swiss-Protnumbering is the same as A1080 in the PDB database and for LCK_HUMANwhere E319 according to Swiss-Prot numbering is the same as E320 by PDBnumbering.

[0122] The ψ₁₁₂ and φ₁₁₃ dihedral angles of the P38γ-AMPPNP complexshown in Table 2 indicate that the conformation of Gly113 in thiscomplex is “flipped” or rotated considerably relative to correspondingamino acids in other MAP kinases. Therefore, the structure coordinatesof P38γ set forth in FIG. 1 represent, inter alia, what is believed tobe a conformation at Met 112 and Gly113 that had not been observed forother crystalline protein kinases, especially other MAP kinases.

[0123] Accordingly, another embodiment of this invention relates to acrystalline protein kinase-ligand complex, said kinase comprising aminoacid residues that correspond by functional and/or sequence alignment tothe Met112 and Gly113 residues of P38γ or that correspond by functionaland/or sequence alignment to the Met112 and Gly113 equivalent residuesof one or more proteins listed in Table 1, wherein the ψ angle of theresidue corresponding to Met112 is in the range of about −60° to 60° andthe φ angle of the residue corresponding to Gly113 is in the range ofabout 30° to 150°. Preferably, the ψ angle of the crystalline proteinkinase-ligand complex is in the range of about −45° to 45° and mostpreferably in the range of about −30° to 30°. Preferably, the φ angle isin the range of about 45° to 135°, and most preferably is in the rangeof about 60° to 120°. Examples of kinases that may provide such acrystalline protein kinase when complexed with a ligand are described byHanks et al., Science, 241, 42 (1988) and Hanks and Quinn, Methods inEnzymology, 200, 38 (1991). Other examples of such kinases may be foundat www.sdsc.edu/Kinases/pkr/pk_catalytic/pk_hanks_seq_align_long.html,where the kinases are listed with their corresponding sequencealignments.

[0124] Another embodiment of this invention relates to a crystallineprotein kinase-ligand complex, said kinase selected from the proteinslisted in Table 1, wherein the ψ angle of the residue corresponding toMet112 is in the range of about −60° to 60° and the φ angle of theresidue corresponding to Gly113 is in the range of about 30° to 150°.Preferably, the ψ angle of the crystalline protein kinase-ligand complexis in the range of about −45° to 45° and most preferably in the range ofabout −30° to 30°. Preferably, the φ angle is in the range of about 45°to 135°, and most preferably is in the range of about 60° to 120°.

[0125] Structural information regarding the conformation of the Met112and Gly113 residues of the crystalline P38γ complex may be encoded in amachine-readable data storage medium as described above for encoding theother structural coordinates of the protein. Accordingly, anotherembodiment of this invention relates to a computer for producing athree-dimensional representation of an ATP binding site of a proteinkinase-ligand complex, or portion thereof, wherein said computercomprises:

[0126] a) a machine-readable data storage medium comprising a datastorage material encoded with machine-readable data, wherein saidmachine-readable data comprises the structure coordinates of a kinase,or portion thereof, said kinase or portion thereof comprising amino acidresidues that correspond by functional and/or sequence alignment to theMet112 and Gly113 residues of P38γ or that correspond by functionaland/or sequence alignment to the Met112 and Gly113 equivalent residuesof one or more proteins listed in Table 1, wherein the ψ angle of theresidue corresponding to Met112 is in the range of about −60° to 60° andthe φ angle of the residue corresponding to Gly113 is in the range ofabout 30° to 150°;

[0127] b) a working memory for storing instructions for processing saidmachine-readable data;

[0128] c) a central-processing unit coupled to said working memory andto said machine-readable data storage medium, for processing saidmachine readable data into said three-dimensional representation; and

[0129] d) an output hardware coupled to said central-processing unit,for receiving said three-dimensional representation. Preferably, themachine-readable data comprises the structure coordinates of a kinase,or portion thereof, said kinase comprising amino acid residuescorresponding to the Met112 and Gly113 amino acids of P38γ orcorresponding to the Met112 and Gly113 equivalent residues of one ormore proteins listed in Table 1, wherein the ψ angle is in the range ofabout −45° to 45° and most preferably in the range of about −30° to 30°,and the φ angle is in the range of about 45° to 135°, and mostpreferably in the range of about 60° to 120°. In a more preferredembodiment of this computer, the machine readable data comprises thestructure coordinates of a crystalline protein kinase-ligand complex, orportion thereof, where said kinase is selected from a protein listed inTable 1.

[0130] For designing new compounds that associate with a protein kinasebinding pocket, it is useful to employ information that includes theconformations of the Met112 and Gly113 residues, or their equivalents,along with other structural information regarding amino acids in thebinding pocket. For example, to evaluate the ability of a chemicalentity to bind to a protein kinase, the conformations of Met112 andGly113, or equivalents, may be used along with structure coordinates ofthe backbone atoms of amino acids in the protein kinase binding pocket.These structure coordinates and the structure coordinates of the p38γamino acids Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110,Phe111, Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157,Asn158, Ala160, Leu170, Asp170, Gly173, and Leu174 according to FIG. 1should not differ by more than about 3.0 angstroms in root mean squaredeviation, preferably the root mean square deviation is within about 2.7angstroms, and most preferably within about 2.5 angstroms. For example,the root mean square deviation between the structure coordinates of thep38γ amino acids and those of a p38γ complex (see Table 2) was found byapplicants to be 2.41 angstroms. Resolution error may account forvariation in the root mean square deviation of a few tenths of anangstrom.

[0131] Accordingly, another embodiment of this invention provides amethod for evaluating the ability of a chemical entity to associate witha protein kinase binding pocket, said method comprising the steps of:

[0132] a) creating a computer model of the binding pocket usingstructure coordinates wherein:

[0133] (i) the root mean square deviation between said structurecoordinates and the structure coordinates of the P38γ amino acids Val33,Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112,Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160,Leu170, Asp170, Gly173, and Leu174 according to FIG. 1 is within about3.0 angstroms,

[0134] (ii) said binding pocket model depicts amino acid residues thatcorrespond by functional and/or sequence alignment to the Met112 andGly113 residues of P38γ or that correspond by functional and/or sequencealignment to the Met112 and Gly113 equivalent residues of one or moreproteins listed in Table 1, and

[0135] (iii) said binding pocket model depicts the ψ angle of theresidue corresponding to Met112 to be in the range of about −60° to 60°and the φ angle of the residue corresponding to Gly113 to be in therange of about 30° to 150°;

[0136] b) employing computational means to perform a fitting operationbetween the chemical entity and the binding pocket model; and

[0137] c) analyzing the results of said fitting operation to quantifythe association between the chemical entity and the binding pocketmodel.

[0138] A useful root mean square deviation between the structurecoordinates of a particular binding pocket and the structure coordinatesof the binding pocket of another protein kinase may be readilydetermined by one skilled in the art. For example, when the proteinkinase is selected from a protein listed in Table 1, the root meansquare deviation is preferably within about 2.7 angstroms, and is morepreferably within about 2.5 angstroms.

[0139] This invention also provides a method for identifying a potentialagonist or antagonist of a molecule comprising a P38γ-like bindingpocket, comprising the steps of:

[0140] a) creating a computer model of the binding pocket usingstructure coordinates wherein:

[0141] (i) the root mean square deviation between said scructurecoordinates and the structure coordinates of the P38γ amino acids Val33,Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112,Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160,Leu170, Asp170, Gly173, and Leu174 according to FIG. 1 is within about3.0 angstroms,

[0142] (ii) said binding pocket model depicts amino acid residues thatcorrespond by functional and/or sequence alignment to the Met112 andGly113 residues of P38γ or that correspond by functional and/or sequencealignment to the Met112 and Gly113 equivalent residues of one or moreproteins listed in Table 1, and

[0143] (iii) said binding pocket model depicts the ψ angle of theresidue corresponding to Met112 to be in the range of about −60° to 60°and the φ angle of the residue corresponding to Gly113 to be in therange of about 30° to 150°;

[0144] b) employing said model of the binding pocket to design or selectsaid potential agonist or antagonist;

[0145] c) synthesizing said agonist or antagonist; and

[0146] d) contacting said agonist or antagonist with said molecule todetermine the ability of said potential agonist or antagonist tointeract with said molecule.

[0147] A preferred embodiment of this method uses the structurecoordinates of the Met112 and Gly113 amino acids of p38γ or the Met112and Gly113 equivalent residues of a protein listed in Table 1.

[0148] In order that this invention be more fully understood, thefollowing examples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

EXAMPLE 1 Expression and Purification of P38γ Protein

[0149] P38 with a His6 tag was overexpressed in E.Coli, and thenpurified by using metal affinity resin followed by MonoQ resin. Thepurified material was phosphorylated with constituitively active MKK6,and purified again with MonoQ resin (Fox, T. et al., manuscript inpreparation). Size-exclusion chromatography was performed to determinethe apparent molecular weights of unphosphorylated and phosphorylatedP38γ as follows. A Superdex 75 HR 10/30 column (Pharmacia, Uppsala) wasequilibrated in 12.5 mM HEPES, pH 7.3, containing 6.25% (v/v) glyceroland 100 mM KCl. Bovine serum albumin (67 kDa), ovalbumin (43 kDa),chymotrypsinogen (25 kDa), ribonuclease A (13.7 kDa) were used tocalibrate the column prior to P38γ analyses. A flow rate of 0.25 ml/minwas used for chromatographic runs and samples were loaded in a volume of100-200_(—)1 at 0.7-4 mg/ml.

EXAMPLE 2 Crystallization of P38γ

[0150] Crystals of phosphorylated P38γ complexed with AMP-PNP were grownby vapor diffusion. Clusters of rods appeared after 3 to 7 days whenprotein (0.5 mM P38γ with 5 mM AMP-PNP and 0.02% C₁₂E₉) was mixed withan equal volume of reservoir (100 mM NaOAc, 100 mM Tris 8.5, 27% PEG4000, 10 mM MgCl₂, and 5 mM DTT) and allowed to stand at roomtemperature. Single crystals with 100 mM maximum thickness wereseparated from their parent cluster, cryoprotected by adding ethyleneglycol to a final concentration of 15% over 15 min in three equal steps,and flash cooled to −170° C. in a stream of gaseous nitrogen.

EXAMPLE 3 X-Ray Data Collection and Structure Determination

[0151] The diffraction pattern displayed symmetry consistent with spacegroup P2₁2₁2₁, with unit cell dimensions a=63.50 Å, b=66.82 Å, andc=206.02 Å. Diffraction extended to 4.0 Å in the a*, b* direction and3.0 Å in the c* direction. Data collection at NSLS X25 allowed asignificant improvement in the observed diffraction limit: data werecollected to 3.0 Å in the a*, b* direction and at least 2.4 Å in the c*direction. Data were integrated to 2.4 Å [Otwinowski, Z. in CCP4 StudyWeekend (eds. Sawyer, L., Isaacs, N. & Bailey, S.) 56-62 (SERC DaresburyLaboratory, England) (1993); Minor, W. XDISPLAYF Program, PurdueUniversity, (1993)]. The overall R-merge for the data was 6.7%, withI/sig (I)=2.0 at 2.4 Å resolution. The X-ray data comprised 31732 uniquereflections derived from 118429 intensity measurements. The data were90% complete overall and 76.5% complete in the 2.49-2.40 Å resolutionshell. Data incompleteness, particularly in the highest resolutionshell, reflects the anisotropic nature of the diffraction.

[0152] The volume of the asymmetric unit indicated the presence of twoP38γ molecules. The self-rotation function calculated with POLARAFN[Acta Crys D50, 760-763 (1994)] revealed a noncrystallographic peak withintensity half of the origin at Kappa=180°, omega=90°, and Phi=44°.

[0153] Coordinates for the structure of phosphorylated ERK2 were notinitially available from the protein data bank and could not be-used formolecular replacement. Several different models for P38γ wereconstructed based on the X-ray coordinates of P38γ or unphosphorylatedERK2 with either all side chains truncated to alanine, or with only thenonconserved side chains truncated to alanine or glycine [Zhang et al.,Nature 367, 704-711 (1994); Wilson and Su, J Biol Chem 271, 27696-27700(1996)]. No rotation function solutions were obtained using these modelswith either the X-plor or AMORE molecular replacement packages. Theanisotropy of the data, as well as the presence of two molecules in theasymmetric unit, could be reasons for the lack of a successful molecularreplacement solution. Variability in the orientation between the largeand small kinase domains may have been an additional complicatingfactor.

[0154] To position correctly an initial P38γ model, experimental phasesat low resolution were obtained from two derivatives. Crystals weresoaked with 0.2 mM ethylmercurychloride (EMP) for 5 days, and with 2 mMEuCl₃ overnight. Diffraction data were collected on the in houseRaxisIIc, and integrated to 5.0 Å [see Owinowski and Minor, supra].Difference Patterson maps were interpreted by using SHELXS-97 [Acta CrysA46, 467-473 (1990)]. The EMP derivative yielded four sites and theEuropium derivative yielded two sites. These heavy atom positions wererefined by using ML-PHARE [Acta Crys D50, 760-763 (1994)] which yieldedan overall figure of merit of 0.53 to 5 Å. The resulting electrondensity maps showed clear solvent and protein regions. Six heavy atomsites were identified within a continuous envelope of protein densityand grouped into two sets of three sites. These two sets were related toone another by a two-fold axis, which was consistent with theself-rotation function. Each set of three sites was assumed tocorrespond to a monomer of P38γ, and the two-fold operation was used toimprove the experimental electron density by noncrystallographicsymmetry (NCS) averaging. Solvent flattening combined with two-foldaveraging using Dm (final correlation coefficient of averaging of 0.851)produced an electron density map at 5.0 Å that allowed placement of theP38γ model. The N-terminal domain had to be rotated by several degreeswith respect to the C-terminal domain in order to fit both domains intothe experimental density. At this stage the model was refined againstthe high resolution synchrotron data. Rigid body refinement andtorsional dynamics refinement yielded an initial R_(free) of 42%.

[0155] The quality of the model was improved by cycles of modelbuilding, positional refinement, and thermal factor refinement,interspersed with torsional dynamics runs using data from 50.0 to 2.4 Å.All stages of model refinement were carried out using the new programCNS [Acta Crys D54, 905-921 (1988)] with bulk solvent correction andanisotropic scaling. NCS restraints were applied throughout therefinement. The current P38γ model contains two monomers, each with 329protein residues, one bound AMP-PNP molecule, and two Mg²⁺ ions. A totalof 186 water molecules were included in the entire asymmetric unit. Thecurrent R_(work) is 23.2% (R_(free)=28.3%) versus all data with|F|>2_(F) between 50-2.4 Å resolution (27841 reflections). PROCHECK wasused to analyze the model stereochemistry [Acta Crys D50, 760-763(1994)]. All of the residues were in the most favored and additionalallowed regions of the Ramachandran plot. One residue per monomer(Val187) from the phosphorylation loop was in the disallowed region. TheP38γ model has deviations from ideal bond lengths and angles of 0.010 Åand 1.63° respectively. No electron density was observed for amino acids1-7, 34-39, 316-321, 330-334, and 354-end, therefore these residues werenot included in the model. The eight residue histidine tag and 21residues at the C-terminus are also disordered. Subsequent to thestructure refinement, the phosphorylated ERK2 coordinates were released,and the final refined P38γ structure was compared with that structure.

EXAMPLE 4 Overall Structure

[0156] The P38γ structure was solved with a combination of lowresolution MIR and molecular replacement using a model of theunphosphorylated form of P38α [Wilson and Su, J Biol Chem 271,27696-27700 (1996)]. The current structure includes two P38γ moleculesper asymmetric unit, each with 329 amino acids, a bound AMP-PNP, and twoMg²⁺ ions. A total of 186 water molecules were modeled in the asymmetricunit. The current R_(free) and R_(work) are 28.3% and 23.2%,respectively. The refined model has deviations from ideal bond lengthsand angles of 0.01 Å and 1.6°. The two P38γ molecules in the asymmetricunit superimpose with an overall r.m.s.d. of 0.013 Å using all Cα atoms,and thus represent two independent but highly similar structures ofactivated P38γ.

Comparison of Kinase Structures

[0157] Electron density for the main chain atoms of P38γ is visible fromresidue 8 to 353, with breaks at residues 34-39, 316-321 and 330-334(FIG. 1). The glycine rich loop, which contains the consensusGly-X-Gly-X-X-Gly sequence (residues 34-39 in P38γ) is mobile, andresidues 34-39 could not be modeled. The homologous region of P38α isalso flexible, and has average B-values equal to 61 Å. In contrast, theAMP-PNP ligand is well ordered, as are all nearby secondary structuralelements. Strong electron density for the residues at the N- andC-terminal ends of the glycine rich loop is also observed. TheC-terminal 40 residues of both P38γ molecules in the asymmetric unit arenot as well ordered as the rest of the structure. Helix αL16 can bemodeled, but contains several disordered side chains. The region justbefore helix αL16 is poorly ordered and does not form the 3/10 helix L16observed in the structure of phosphorylated ERK2. Helix αL16 and 3/10helix L16 are involved in dimer formation in the structure ofphosphorylated ERK2 [Canagarajah et al., Cell 90, 859-869 (1997)].

[0158] Activated P38γ contains a small amino terminal domain comprisedmainly of β-strands, and a large carboxyl terminal domain that consistsmostly of α-helices (FIG. 1). This fold is common among kinases [Taylor& Radzio-Andzlem (1994); Structure 2, 345-355; Kultz J Mol Evol 46,571-588 (1998)]. A deep cleft at the interface between the domains formsthe binding site tor ATP and Mg²⁺. The two domains are connected by ahinge, located at a point adjacent to the adenine base and near residue113 (FIG. 1).

[0159] Whereas the sequence, fold, and topology of P38γ is similar toP38α (FIGS. 1, 2), the domains of activated P38γ are closed relative toP38α Independent superimpositions of the domains of P38γ onto the P38αstructure yield r.m.s. deviations of 1.2 Å for the N-terminal domain(P38γ Cα carbons from residues 10-16, 19-33, and 40-113), and 0.62 Å forthe C-terminal domain (P38γ C_(α) carbons from residues 125 to 160, 206to 238 and 282 to 297). Greater differences between P38γ and P38α areobserved when the whole proteins are compared. Superimposition of theC-terminal domain of P38γ onto the corresponding lobe of P38α revealed arotation of the N-terminal domain of P38γ by 20° relative to theorientation seen in P38α (FIG. 2). Other differences between thestructure of phosphorylated P38γ and P38α occur in the conformation ofα1L14, α2L14, α1L12, the phosphorylation loop, and αL16.

[0160] Inter-domain rotation, or domain closure, is common in MAP kinasestructures, and is observed to different extents. The structures ofunphosphorylated and phosphorylated ERK2 show a 5° difference in domainclosure. The structure of unphosphorylated JNK3 reveals that a 10°domain rotation would be needed to superimpose both domains with thestructure of phosphorylated P38γ or phosphorylated ERK2. P38α MAP kinaseis more open in its unphosphorylated state than ERK2 or JNK3. Despite alarge difference in the conformations of the unphosphorylated proteins,the domains of the activated forms of P38γ and ERK2 can be superimposedwith a rotation of only 3°. Comparison to solved kinase structuresindicates that the relative positions of the domains in activated P38γis most similar to activated ERK2 MAP kinase.

[0161] The structures of phosphorylated P38γ and phosphorylated ERK2 aresimilar, with a few significant differences. One conformationaldifference is a movement of the α1L14, α2L14 helical region. With thelarge domains superimposed, the difference in α1L14, α2L14 orientationbetween the two structures is about 6 Å, when measured at the mostextreme portion of the helices. Another difference between the twostructures is that the P38γ activation loop is six residues shorter thanthe activation loop in ERK2. Excluding these two regions allows one tosuperimpose P38γ Cα carbons 19-33, 40-58, 61-94, 97-113, 117-177,182-243, and 269-315 with the corresponding ERK2-P2 atoms to yield anr.m.s.d. of 1.1 Å. This reflects the high similarity between the twostructures. A comparison of the activation loops, using P38γ Cα carbons173-177 and 182-188 yields an r.m.s.d. of 0.3 Å.

[0162] The structure of the phosphorylation loop differs betweenphosphorylated P38γ and unphosphorylated P38α (FIG. 2). Thephosphorylation loop contains the TGY sequence present in all P38 MAPkinases. Phosphorylation of Thr183 and Tyr185 results in a movement ofthe activation loop, and produces changes in the P38γ structure.

[0163] Phospho-Thr183 sits at the interface between the two domains. TheThr183 phosphate group interacts with Arg70, Arg73 and Lys69 from theN-terminal domain, and Arg152 and Arg176 from the C-terminal domain(FIGS. 1 and a point adjacent to the adenine base and near residue 113.The hinge-point and residue pThr183 are located at opposite ends of theinterface between the two domains. The network of interactions betweenpThr183 and these basic residues pulls the domains together. As aresult, the relative orientations of the amino acids, including thecatalytic residues, located between the hinge and pThr183 are changed. Asimilar set of interactions between the phospho-threonine and nearbybasic residues was reported for the structure of phosphorylated ERK2[Canagarajah et al., Cell 90, 859-869 (1997)]. Phosphorylated P38γ is ina conformation consistent with activity. The active site ofphosphorylated P38γ is shown in detail in FIG. 4, and compared with theactive sites of P38α and cAPK in FIGS., 5 a and 5 b. The interactionsbetween the non-hydrolyzable nucleotide analog AMP-PNP and P38γ (FIG. 4)are very similar to those made between bound nucleotide and cAPK [Zhenget al., Acta Cryst. D49, 362-365 (1993); Bossemeyer et al., EMBO Journal12, 849-859 (1993); Narayana et al., Structure 5, 921-935 (1997)].

[0164] The N1 and N6 nitrogen atoms of AMP-PNP form hydrogen bonds tothe backbone amide nitrogen atom of Met112 and the backbone carbonyloxygen atom of Pro110, respectively. Interactions between the glycinerich loop and the nucleotide are not observed in the P38γ structure. Therelative positions of catalytic residues Lys53, Glu74 and Asp153 provideinformation about the state of activation of the kinase [Kumar et al.,J. Biol. Chem. 270, 29043-29046 (1995); Robinson et al., Curr Opin CellBiol 9, 180-186 (1997)]. Comparison of P38γ with cAPK aftersuperimposing the nucleotides from the two structures (FIG. 5b), revealsthat the active site residues in the two structures are in almost thesame conformation. The cAPK structure also contains a bound peptideinhibitor, and the complex is believed to represent a bioactiveconformation of cAPK [Zheng et al, supra; Bossemyer et al., supra;Narayana et al., supra]. The nucleotides in both structures adopt almostthe same conformations, and the relative positions of the catalyticresidues Lys-56, Glu-74 and Asp-153 are conserved. There are also twobound metal ions in each complex. After superimposition, metal I in cAPKis separated from the corresponding metal in P38α by 0.5 Å, and metal IIfrom P38γ is 1.4 Å removed from metal II in cAPK. Because theconformation and relative orientation of the catalytic residues andcofactors in the active sites of the two kinases are almost the same,the structure of phosphorylated P38γ described here is likely torepresent an active conformation.

[0165] Comparing the phosphorylated P38γ to the known, unphosphorylatedP38α one finds that the active site residues of P38α are significantlydisplaced relative to their orientation in P38γ. This presumablyreflects the inactive state of unphosphorylated P38α (FIG. 5a). Twotypes of structural differences are observed between unactivated P38αand activated P38γ. A rigid body motion occurs between the two domains,and secondary structure elements and residues move as a result ofphosphorylation and AMP-PNP binding.

[0166] Using the newly-determined structure of P38γ, the structure ofunphosphorylated P38α could be altered to properly position itscatalytic residues in an active conformation. Without the P38γstructural information, it was not known whether domain movement alonewould be enough to properly position the catalytic in an activeconformation or whether activation would also require other changes[Johnson et al., Curr. Opin. Struct. Biol. 6, 762-769 (1996); Yamaguchiet al., Nature 384, 484-489 (1996); Johnson et al., Cell 85, 149-158(1996); Russo et al., Nature Struc Biol 3, 696-700 (1996)].

[0167] To address this question, the structure of unphosphorylated P38αwas altered to resemble phosphorylated P38γ. Only a rigid-body movement,centered on the hinge residue 113, was used to change the relativeorientation of the two domains in P38α. The resulting model maintainsthe detailed secondary structure features present in non-phosphorylatedP38α, but has the same domain closure as P38γ. The positions ofcatalytic residues in the active site of this modified P38γ model matchwell to those observed in the structure of activated P38γ. The rigidbody movement shifts P38α residue Lys-53 2.9 Å closer to its counterpartin P38γ (from 4.4 Å to 1.5 Å separation). Glu-71 (P38α) moves 2.8 Ånearer to its equivalent residue in P38γ (from 3.2 Å to 0.4 Åseparation). Thus, the structures of P38α and P38γ suggest that a simpledomain rotation accounts for most of the rearrangement of catalyticresidues necessary for activation of P38γ.

[0168] Other movements may contribute to activation of P38γ. Forexample, phosphorylation of Tyr185 leads to a rearrangement ofsurrounding secondary structure elements that may effect substratebinding. Arg192 interacts with the pTyr185 phosphate group in the P38γstructure, and is shifted more than 5 Å relative to its position in theapo-P38α structure. Such coordination of Arg 192 and its effect onsubstrate binding have been discussed with regard to ERK2and JKN3[Zhang, Nature 367, 704-711 (1994); Xie and Su, supra; Canagarajah,supra]. In the P38γ structure, pTyr185 interacts directly with Arg189and Arg192 (FIG. 3). Comparison of the P38γ pTyr185 conformation, aswell as the backbone conformation with the corresponding residue ofphosphorylated ERK2, shows that the two residues are in nearly the sameconformation.

Activated P38_is Monomeric

[0169] The two P38γ proteins in the crystallized complex show noevidence of dimeric interaction, as evidenced by the examination of theactivation loops of the two proteins. This is unlike the activated,phosphorylated ERK2, which is believed to reveal a dimer interface thatis not observed in the non-phosphorylated form [Zhang et al., supra;Canagarajah et al., supra; Khokhlatchev et al., supra]. The dimerinterface in phosphorylated ERK2 reportedly buries a total of 1470 Å² ofsurface area, and is formed in part by an ion pair between His176 fromone molecule and Glu343 from the other molecule. In addition, Leu333,Leu336, and Leu344 are reported to further stabilize the dimerinterface.

[0170] The entire surface of each P38γ molecule in the asymmetric unitwas examined in search of any dimer interface. The crystal of P38γbelongs to space group P2₁2₁2₁, which contains only two-fold screw axes,but no crystallographic two-fold axes. The only two-fold axis in thecrystal is the non-crystallographic axis which relates the two moleculeswithin the asymmetric unit. This dimeric interaction involves Pro282,Asn286, Lys290, Leu283, Pro309, and Glu312. This non-crystallographicdimer interface buries only 680 Å² of surface area, less than half ofthe 1470 Å² buried in the phosphorylated ERK2 dimer interface.

[0171] To characterize further the oligomeric state of activated P38γ insolution, size-exclusion chromatography was performed to determine theapparent molecular weights of unphosphorylated and phosphorylated P38γ.To facilitate comparison with the phosphorylated ERK2 results[Khokhlatchev et al., supra], the same column resin, buffer, and loadingconditions were used. The chromatographic profiles of unphosphorylatedand phosphorylated P38γ showed that both proteins eluted with a similarretention time, corresponding to a molecular. weight of 44.5 kDa asdetermined from the protein calibration curve. The absence of dimerformation of phosphorylated P38γ in solution is consistent with theabsence of dimer formation in the crystal structure of P38γ. It is alsoconsistent with the absence of dimer formation in ERK2 mutants whereHis176 is deleted [Khokhlatchev et al., supra].

[0172] Conformations of Activation Loops of Kinases

[0173] The number of residues in the activation loops of differentkinases varies, ranging from 8 amino acids in calmodulin dependentDAP-kinase to 37 residues in LIMK2 [Deiss et al., Genes Dev. 9, 15-30(1995); Okano et al., J. Biol. Chem. 270, 31321-31330 (1995)]. The P38γactivation loop consists of residues Gly173-Thr188. The phosphorylationloop of ERK2 is six residues longer in sequence and spans amino acidsGly167-Thr188. The loop region of cAPK is the same length as P38γ, andspans amino acids Gly186-Thr201. FIG. 6 highlights the loop regions fromP38γ, ERK2-P2, and cAPK. Except for a longer loop size for ERK2, thestructures of the loop regions of activated P38γ and activated ERK2arenearly identical. The distance between the phosphate moieties fromThr183 in P38γ and ERK2 is only 0.4 Å, and separation between the Tyr185phosphate from P38γ and ERK2 is 1.6 Å. The phosphorylation loop of cAPKdoes not superimpose as well with the two MAP kinase phosphorylationloops, although the Thr phosphate is only 2.0 Å away from the P38γThr183 phosphate. The phosphorylation loop regions from P38γ, ERK2 andcAPK have different lengths, but in their phosphorylated states adoptalmost the same conformations.

[0174]FIGS. 1a-6 further depict the structure of the phosphorylatedP38γ/MgAMP-PNP complex. Thus, FIG. 1a depicts an overview of thephosphorylated P38γ structure. The large and small domains are pulledtogether by interactions mediated by phospho-Thr183. Ribbon diagrams ofthe activated P38γ structure with the amino-terminal small domain arecolored light orange and the carboxy-terminal large domain colored blue.The interface between the two domains (residue 113) can be thought of asa hinge point through which domain movement occurs. Four Arg residuesand one Lys residue are explicitly shown coordinated to the phosphate ofpThr183. Arg70, Arg73 and Lys69 anchor the small domain to pThr183, andArg152 and Arg176 anchor the large domain to pThr183. PThr183 pulls thedomains together. All figures were made with RIBBONS [Carson et al., J.Mol. Graphics 4, 121-122 (1986)].

[0175]FIG. 2 is a superimposition of the structures of unphosphorylatedP38α and phosphorylated P38γ. P38α is shown in light blue and dark blue(activation loop), and P38γ is shown in light orange and dark orange(activation loop). The Cα atoms from residues 125 to 160, 206 to 238 and282 to 297 were used to superimpose the two proteins with an r.m.s.d. of0.62 Å. Also shown is the AMP-PNP and two Mg²⁺ ions from the P38γstructure. All atoms of the phosphorylated Thr183 and Tyr185 from theP38γ structure are shown. Major changes upon phosphorylation are asignificant domain closure and a rearrangement of the activation loop.

[0176]FIG. 3 is a detailed stereo view of activation loop. All atomstereo view of the P38γ activation loop (residues 174 to 189). Residuesthat coordinate pThr183 and pTyr185 are also shown. Hydrogen bonds areindicated with dashed grey lines. The phosphate atoms are shown in pink.

[0177]FIG. 4 is a stereo view of AMP-PNP. All major interactions withprotein side chains are indicated with dashed grey lines. The bound Mg²⁺ions are indicated by black spheres. The phosphate atoms are shown inpink. Met109 can be seen behind the adenine base, blocking thehydrophobic pocket. Water molecules have been removed for clarity.

[0178]FIGS. 5a and 5 b are a comparison of the active site of activatedP38γ with P38α and cAPK. P38γ is shown in orange, P38α in blue, and cAPKin red. In all three structures a salt bridge is observed between Lys56and Glu74 (P38γ numbering). a) Comparison of the active sites of P38γwith P38α by superimposition of their carboxyl terminal large domains.Catalytic residues are misaligned. The distance between Asp153 and Lys53is 12.6 Å in the P38α structure compared with 8.5 Å in thephosphorylated P38γ structure. b) Comparison of the active sites of P38γwith cAPK (Protein Data Base code: 1ATP, ref. 22) by superimrposition ofall atoms of their bound AMP-PNP molecules. All catalytic residues alignto within a fraction of an Å. The distance between Asp153 and Lys53 is8.5 Å in the activated P38γ structure. This distance is very close tothe distance of 7.8 Å observed in activated cAPK, suggesting that thestructure reported here is of the activated kinase. Asp171 is excludedfrom these figures for clarity because it is obscured by AMP-PNP andMg²⁺ ions.

[0179]FIG. 6 is a comparison of activated phosphorylation loops fromP38γ (dark orange), ERK2 (dark blue), and cAPk (red). Superimposition ofthese three structures was with the C_(α) atoms of residues 125 to 160,206 to 238 and 282 to 297 of P38γ.In order to ensure an unbiasedcomparison of the lip regions, these residues were omitted from thecalculation. All three lip regions have different lengths, but havesurprisingly similar conformation. Comparison of P38γ and ERK2superimposes the two phorphorylated amino acids almost exactly, despitea six amino acid difference in length. The phosphorylated Thr197 of cAPKalso superimposes well with the two MAP kinase structures. Thiscomparison suggests that the phosphorylated lip structures observed inP38γ and ERK2 may be representative of all MAP kinases.

EXAMPLE 5 The Use of P38γ/MgAMP-PNP Coordinates for Inhibitor Design

[0180] The coordinates of FIG. 1 are used to design compounds, includinginhibitory compounds, that associate with P38γ or homologues of P38γ.This process may be aided by using a computer comprising amachine-readable data storage medium encoded with a set ofmachine-executable instructions, wherein the recorded instructions arecapable of displaying a three-dimensional representation of theP38γ/MGAMP-PNP complex or a portion thereof. The graphicalrepresentation is used according to the methods described herein todesign compounds. Such compounds associate with the P38γ at the activesite.

EXAMPLE 6 P38γ Activity Inhibition Assay

[0181] To determine the IC₅₀ of compound binding to P38γ, the kinaseactivity of P38Y was monitored by coupled enzyme assay. In this assay,for every molecule of ADP generated by the P38Y kinase activity onemolecule of NADH is converted to NAD which can be conveniently monitoredas an absorbance decrease at 340 nm. The following are the finalconcentrations of various reagents used in the assay: 100 mM HEPESbuffer, pH 7.6, 10 mM MgCl₂, 30 μM ATP, 2 mM phosphoenolpyruvate, 2 μMpyruvate kinase, 2 μM lactate dehydrogenase, 200 μM NADH, 200 μM EGFreceptor peptide KRELVEPLTPSGEAPNQALLR, and 10 nM activated P38γ. First,all of the above reagents with the exception of ATP were mixed and 175μl aliquots were placed per well of 96-well plate. A 5 μl DMSO solutionof the compound was added to each well, mixed, and allowed to stand at30° C. for 10 minutes. Typically about 10 different concentrations ofthe compound were tested. The reactions were initiated with the additionof 20 μl of ATP solution. Absorbance change at 340 nm were monitored asa function of time. IC₅₀ is obtained by fitting the rates vs. compoundconcentration data to a simple competitive inhibition model.

[0182] While we have described a number of embodiments of thisinvention, it is apparent that our basic constructions may be altered toprovide other embodiments which utilize the products, processes andmethods of this invention. Therefore, it will be appreciated that thescope of this invention is to be defined by the appended claims, ratherthan by the specific embodiments which have been presented by way ofexample.

We claim:
 1. A crystalline composition comprising a phosphorylated P38protein-ligand complex.
 2. The crystalline composition of claim 1wherein the complex is capable of being resolved at 2.4 Å resolution,the complex comprising: a) a purified enzyme selected fromphosphorylated P38α, phosphorylated P38β, phosphorylated P38δ,phosphorylated P38γ, or a phosphorylated isoform of any of theforegoing; b) a ligand; and c) magnesium ions.
 3. The crystallinecomposition according to claim 2, wherein said enzyme is P38γ.
 4. Acrystalline protein kinase-ligand complex, said kinase comprising abinding pocket defined by the structure coordinates of the P38γ aminoacids Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111,Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158,Ala160, Leu170, Asp171, Gly173, and Leu174 according to FIG. 1, or ahomologue of said kinase, wherein said homologue comprises a bindingpocket that has a root mean square deviation from the backbone atoms ofsaid amino acids of not more than 1.15 Å.
 5. A crystalline proteinkinase-ligand complex, said kinase selected from the proteins listed inTable 1, wherein the ψ angle of the residue corresponding to Met112 ofp38γ is in the range of about −60° to 60° and the φ angle of the residuecorresponding to Gly113 of p38γ is in the range of about 30° to 150°. 6.The crystalline protein kinase-ligand complex of claim 5 wherein the ψangle is in the range of about −45° to 45°.
 7. The crystalline proteinkinase-ligand complex of claim 6 wherein the ψ angle is in the range ofabout −30° to 30°.
 8. The crystalline protein kinase-ligand complex ofany of claims 5 to 7 wherein the φ angle is in the range of about 45° to135°.
 9. The crystalline protein kinase-ligand complex of any of claims5 to 7 wherein the φ angle is in the range of about 60° to 120°.
 10. Acrystalline protein kinase-ligand complex, said kinase comprising aminoacid residues that correspond by functional and/or sequence alignment tothe Met112 and Gly113 residues of P38γ or that correspond by functionaland/or sequence alignment to the Met112 and Gly113 equivalent residuesof one or more proteins listed in Table 1, wherein the ψ angle of theresidue corresponding to Met112 is in the range of about −60° to 60° andthe φ angle of the residue corresponding to Gly113 is in the range ofabout 30° to 150°.
 11. A method for evaluating the ability of a chemicalentity to associate with a molecule or molecular complex comprising abinding pocket, said method comprising the steps of: a) creating acomputer model of the binding pocket using structure coordinates whereinthe root mean square deviation between said structure coordinates andthe structure coordinates of the P38γ amino acids Val33, Ala40, Val41,Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112, Gly113, Thr114,Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160, Leu170, Asp171,Gly173, and Leu174 according to FIG. 1 is not more than about 1.15 Å; b)employing computational means to perform a fitting operation between thechemical entity and said computer model of the binding pocket; and c)analyzing the results of said fitting operation to quantify theassociation between the chemical entity and the binding pocket model.12. The method according to claim 11, wherein said binding pocket isfurther defined by the structure coordinates of P38γ amino acids Pro32,Cys42, Ser43, Val53, Ile55, Lys57, Leu58, Thr59, Arg70, Glu74, Gly88,Leu107, Val108, Leu116, Gly117, Pro156, Leu159, Val161, Lys168, Phe172,Ala175, and Thr188 according to FIG.
 1. 13. The method according toclaim 12 wherein said molecule or molecular complex is defined by theset of structure coordinates for all P38γ amino acids according toFIG.
 1. 14. A method of utilizing molecular replacement to obtainstructural information about a molecule or a molecular complex ofunknown structure, comprising the steps of: a. crystallizing saidmolecule or molecular complex; b. generating an X-ray diffractionpattern from said crystallized molecule or molecular complex; c.applying at least a portion of the structure coordinates set forth inFIG. 1 to the X-ray diffraction pattern to generate a three-dimensionalelectron density map of at least a portion of the molecule or molecularcomplex whose structure is unknown.
 15. A computer for producing athree-dimensional representation of a molecule or molecular complex,wherein said computer comprises: a) a machine-readable data storagemedium comprising a data storage material encoded with machine-readabledata, wherein said machine-readable data comprises the structurecoordinates of P38γ amino acids Val33, Ala40, Val41, Ala54, Lys56,Ile87, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Lys118,Asp153, Lys155, Gly157, Asn158, Ala160, Leu170, Asp171, Gly173, andLeu174 according to FIG. 1, or structural coordinates having a root meansquare deviation from the backbone atoms of said amino acids of not morethan 1.15 Å; b) a working memory for storing instructions for processingsaid machine-readable data; c) a central-processing unit coupled to saidworking memory and to said machine-readable data storage medium, forprocessing said machine readable data into said three-dimensionalrepresentation; and d) an output hardware coupled to saidcentral-processing unit, for receiving said three-dimensionalrepresentation.
 16. A method for identifying a potential agonist orantagonist of a molecule comprising a P38γ-like binding pocket,comprising the steps of: a. using the atomic coordinates of Val33,Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112,Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160,Leu170, Asp171, Gly173, and Leu174 according to FIG. 1±a root meansquare deviation from the backbone atoms of said amino acids of not morethan 1.15 Å, to generate a three-dimensional structure of moleculecomprising the P38γ-like binding pocket; b. employing saidthree-dimensional structure to design or select said potential agonistor antagonist; c. synthesizing said agonist or antagonist; and d.contacting said agonist or antagonist with said molecule to determinethe ability of said potential agonist or antagonist to interact withsaid molecule.
 17. The method according to claim 16, wherein the atomiccoordinates of Pro32, Val33, Ala40, Val41, Cys42, Ser43, Val53, Ala54,Ile55, Lys56, Lys57, Leu58, Thr59, Arg70, Glu74, Ile87, Gly88, Leu107,Val108, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Leu116,Gly117, Lys118, Asp153, Lys155, Pro156, Gly157, Asn158, Leu159, Ala160,Val161, Lys168, Leu170, Asp171, Phe172, Gly173, Leu174, Ala175, andThr188 according to FIG. 1±a root mean square deviation from thebackbone atoms of said amino acids of not more than 1.15 Å, are used togenerate said three-dimensional structure of the molecule comprising aP38γ-like binding pocket.
 18. The method according to claim 17, whereinthe atomic coordinates of all the amino acids of P38γ according to FIG.1±a root mean square deviation from the backbone atoms of said aminoacids of not more than 1.15 Å, are used to generate a three-dimensionalstructure of molecule comprising a P38γ-like binding pocket.
 19. Acomputer for producing a three-dimensional representation of a proteinkinase or a protein kinase-ligand complex, or portion thereof, whereinsaid computer comprises: a) a machine-readable data storage mediumcomprising a data storage material encoded with machine-readable data,wherein said machine-readable data comprises the structure coordinatesof said kinase, or portion thereof, said kinase or portion thereofcomprising amino acid residues that correspond by functional and/orsequence alignment to the Met112 and Gly113 residues of P38γ or thatcorrespond by functional and/or sequence alignment to the Met112 andGly113 equivalent residues of one or more proteins listed in Table 1,wherein the ψ angle of the residue corresponding to Met112 is in therange of about −60° to 60° and the φ angle of the residue correspondingto Gly113 is in the range of about 30° to 150°; b) a working memory forstoring instructions for processing said machine-readable data; c) acentral-processing unit coupled to said working memory and to saidmachine-readable data storage medium, for processing said machinereadable data into said three-dimensional representation; and d) anoutput hardware coupled to said central-processing unit, for receivingsaid three-dimensional representation.
 20. The computer of claim 19wherein the ψ angle is in the range of about −45° to 45°.
 21. Thecomputer of claim 20 wherein the ψ angle is in the range of about −30°to 30°.
 22. The computer of any of claims 19 to 21 wherein the φ angleis in the range of about 45° to 135°.
 23. The computer of any of claims19 to 21 wherein the φ angle is in the range of about 60° to 120°. 24.The computer of claim 19 wherein the machine-readable data comprises thestructure coordinates of a protein kinase, or portion thereof, saidkinase selected from a protein listed in Table
 1. 25. A method forevaluating the ability of a chemical entity to associate with a proteinkinase binding pocket, said method comprising the steps of: a) creatinga computer model of the binding pocket using structure coordinateswherein: (i) the root mean square deviation between said structurecoordinates and the structure coordinates of the P38γ amino acids Val33,Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112,Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160,Leu170, Asp171, Gly173, and Leu174 according to FIG. 1 is within about3.0 angstroms, (ii) said binding pocket model depicts amino acidresidues that correspond by functional and/or sequence alignment to theMet112 and Gly113 residues of P38γ or that correspond by functionaland/or sequence alignment to the Met112 and Gly113 equivalent residuesof one or more proteins listed in Table 1, and (iii) said binding pocketmodel depicts the ψ angle of the residue corresponding to Met112 to bein the range of about −60° to 60° and the φ angle of the residuecorresponding to Gly113 to be in the range of about 30° to 150°; b)employing computational means to perform a fitting operation between thechemical entity and the binding pocket model; and c) analyzing theresults of said fitting operation to quantify the association betweenthe chemical entity and the binding pocket model.
 26. The method ofclaim 25 wherein the root mean square deviation is within about 2.7angstroms.
 27. The method of claim 26 wherein the root mean squaredeviation is within about 2.5 angstroms.
 28. The method of claim 25wherein the ψ angle is in the range of about −45° to 45°.
 29. The methodof claim 26 wherein the ψ angle is in the range of about −45° to 45°.30. The method of claim 27 wherein the ψ angle is in the range of about−45° to 45°.
 31. The method of claim 25 wherein the ψ angle is in therange of about −30° to 30°.
 32. The method of claim 26 wherein the ψangle is in the range of about −30° to 30°.
 33. The method of claim 27wherein the ψ angle is in the range of about −30° to 30°.
 34. The methodof any of claims 25 to 33 wherein the φ angle is in the range of about45° to 135°.
 35. The method of claim 25 to 33 wherein the φ angle is inthe range of about 60° to 120°.
 36. The method of claim 25 wherein theprotein kinase is selected from a Table 1 protein.
 37. A method foridentifying a potential agonist or antagonist of a molecule comprising aP38γ-like binding pocket, comprising the steps of: a) creating acomputer model of the binding pocket using structure coordinateswherein: (i) the root mean square deviation between said structurecoordinates and the structure coordinates of the P38γ amino acids Val33,Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112,Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160,Leu170, Asp171, Gly173, and Leu174 according to FIG. 1 is within about3.0 angstroms, (ii) said binding pocket model depicts amino acidresidues that correspond by functional and/or sequence alignment to theMet112 and Gly113 residues of P38γ or that correspond by functionaland/or sequence alignment to the Met112 and Gly113 equivalent residuesof one or more proteins listed in Table 1, and (iii) said binding pocketmodel depicts the ψ angle of the residue corresponding to Met112 to bein the range of about −60° to 60° and the φ angle of the residuecorresponding to Gly113 to be in the range of about 30° to 150°; b)employing said model of the binding pocket to design or select saidpotential agonist or antagonist; c) synthesizing said agonist orantagonist; and d) contacting said agonist or antagonist with saidmolecule to determine the ability of said potential agonist orantagonist to interact with said molecule.
 38. The method of claim 37wherein the root mean square deviation is within about 2.7 angstroms.39. The method of claim 38 wherein the root mean square deviation iswithin about 2.5 angstroms.
 40. The method of claim 37 wherein the ψangle is in the range of about −45° to 45°.
 41. The method of claim 38wherein the ψ angle is in the range of about −45° to 45°.
 42. The methodof claim 39 wherein the ψ angle is in the range of about −45° to 45°.43. The method of any of claims 37 to 42 wherein the ψ angle is in therange of about −30° to 30°.
 44. The method of any of claims 37 to 42wherein the φ angle is in the range of about 45° to 135°.
 45. The methodof any of claims 37 to 42 wherein the φ angle is in the range of about60° to 120°.